Table 1. Definitions and data parameters from CyteSeer®’s Lipid Droplet and Colocalization algorithms.

Image channels and masks are abbreviated with two characters, where first letter refers to the cell structure being considered and the second letter (lower case) designates image (i) or mask (m) respectively; thus “Ni” is the nuclear image, whereas “Nm” is the nuclear mask. Area data (Ar) has units of µm²/cell. Lipid droplet count (LDC) is per cell. Average and median pixel intensity (Api and Mpi) values ranged from 0 to 255, as the images in this study were 8-bit depth. Total integrated intensity is the sum of intensities for all pixels in the designated image and mask. For colocalization, L_i and L_aver refer to the individual-, and average-pixel intensities of the lipid image whereas L_i,coloc is the pixel intensity within the lipid image for pixels that are also above threshold in the protein image. Similarly, P_i, and P_aver, refer to the individual-, and average- pixel intensities of the protein image and P_i,coloc, is pixel intensity within the protein image for pixels that are also above threshold in the lipid image. Pearson’s Correlation (P_r) and the Manders’ colocalization coefficients (M₁ and M₂) are dimensionless. P_r ranges from −1.0 (perfect exclusion) to 1.0 (perfect colocalization) with a value of 0 indicating a random distribution between the two labels; M₁ and M₂ range from 0 to 1.0.

Image channels:	Masks:	Area and lipid droplets:
Ni = Nuclear image (DAPI)	Nm = Nuclear	Ar Wm = Area Whole cell mask
Li = Lipid image	Wm = Whole cell	Ar Lm = Area Lipid mask
Pi = Protein image	Cm = Cytoplasm	Ar Pm = Area Protein mask
	Lm = Lipid	LDC = Lipid Droplet Count
	Pm = Protein	LDD = Lipid Droplet Diameter
Pixel Intensity :	Colocalization:
Tii = Total integrated pixel intensity Api = Average pixel intensity Mpi = Median pixel intensity	$\text{Pearson}’{s,\mathrm{P}}_{\mathrm{r}}=\frac{{\displaystyle \sum _i(L_i-L_{\mathit{\text{aver}}})*(P_i-P_{\mathit{\text{aver}}})}}{\sqrt{({\displaystyle \sum _i{(L_i-L_{\mathit{\text{aver}}})}^2*{\displaystyle \sum _i{(P_i-P_{\mathit{\text{aver}}})}^2}})}}$
	$\text{Manders}’{,\mathrm{M}}_1=\frac{{\displaystyle \sum _i{L}_{i,\mathit{\text{coloc}}}}}{\sqrt{{\displaystyle \sum _i{L}_i}}},{\mathrm{M}}_2=\frac{{\displaystyle \sum _i{P}_{i,\mathit{\text{coloc}}}}}{{\displaystyle \sum _i{P}_i}}$
Commonlv used data parameters:
Tii Li Lm = Total integrated pixel intensity, Lipid image, Lipid mask
Api Li Lm = Average integrated pixel intensity, Lipid image, Lipid mask
Mpi Li Lm = Median pixel intensity, Lipid image, Lipid mask.
Pr Li Pi Wm = Pearson’s correlation, Lipid image, Protein image, Whole cell mask
M1 Li Pi Wm = Manders’ M1, Lipid Image, Protein image, Whole cell mask
M2 Li Pi Wm = Manders’ M2, Lipid Image, Protein image, Whole cell mask